In diverse eukaryotes, double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the double-strand (Hutvdgner et al. (2002) Curr. Opin. Genet. Dev. 12:225-232; Hannon (2002) Nature 418:244-25 1). In animals and basal eukaryotes, this process is called RNA interference (RNAi) (Fire et al. (1998) Nature 391:806-811). There is now wide agreement that RNAi is initiated by the conversion of dsRNA into 21-23 nucleotide fragments by the multi-domain RNase III enzyme, Dicer (Bernstein et al. (2001) Nature 409:363-366; Billy et al. (2001) Proc. Natl. Acad. Sci. USA 98:14428-14433; Grishok et al. (2001) Cell 106:23-34; Ketting et al. (2001) Genes Dev. 15:2654-2659; Knight et al. (2001) Science 293:2269-2271; and Martens et al. (2002) Cell 13:445-453). These short RNAs are known as small interfering RNAs (siRNAs), and they direct the degradation of target RNAs complementary to the siRNA sequence (Zamore et al. (2000) Cell 101:25-33; Elbashir et al. (2001) Nature 411:494498; Elbashir et al. (2001) Genes Dev. 15:188-200; Elbashir et al. (2001) EMBO J 20:6877-6888; Nykdnen et al. (2001) Cell 107:309-321; and Elbashir et al. (2002) Clin. Pharmacol. 26:199-213).
siRNA molecules typically have 2- to 3-nucleotide 3′-overhanging ends, which permits them to be capable of interacting with an endonuclease complex, which results in a targeted mRNA cleavage. The potential therapeutic use of siRNA has been demonstrated in a number of systems. RNAi technology has been utilized to successfully target various genes, including HIV rev genes, CD4 and CD8 genes, and P53 genes (Lee, N. S. et al. (2002) Nature Biotechnol. 20: 500-505; Brummelkamp, T. R., et al. Science 2002. 296: 550-553.).
siRNAs have been used in a number of different experimental settings to silence gene expression. For example, chemically synthesized or in vivo transcribed siRNAs have been transfected into cells, injected into mice, or introduced into plants (e.g. by a particle gun). Additionally, siRNAs have been expressed endogenously from siRNA expression vectors or PCR products in cells or in transgenic animals. Besides being utilized for gene silencing, siRNAs have been determined to play diverse biological functions in vivo. This includes roles that include antiviral defense, transposon silencing, gene regulation, centromeric silencing, and genomic rearrangements. Such functional diversity has exemplified the importance of siRNAs within cells and has also stirred interest in their detection across species and tissues. Gene Specific Silencing by RNAi, Tech Notes 10(1). McManus M T and Sharp P A (2002) Gene silencing in mammals by small interfering RNAs. Nature Rev Genet 3: 737-747. Dillin A (2003) Proc Natl Acad Sci USA 100(11): 6289-6291. Tuschl T (2002) Nature Biotechnol 20: 446-448.
An obstacle to the realization of the full potential of gene therapy is the development of safe and effective means for delivering siRNA to cells and organisms. The use of antisense oligonucleotides as therapeutic agents has also been widely investigated in the past few years. Gould-Fogerite et al. Cochleate Delivery Vehicles: Applications to Gene Therapy. Drug Delivery Technology, Vol 3:40-47, 2003. Parker et al. In Vivo and in vitro anti-proliferative effects of antisense IL-10 Oligonucleotides in Antisense Technology, Part B, M. Ian Phillips, Ed., Methods in Enzymology, Vol 314, pp 411-429, 1999; Mannino et al., New Generation Vaccines: “Antigen cochleate formulations for oral and systemic vaccination,” p. 1-9 (Marcel Dekker, New York, 2nd ed. 1997); Brent et al., Neurosci 114(2): 275-278 (2002); Akhtar et al., Nucleic Acids Res. 19:5551 (1991). Their efficacy is based on their ability to recognize their mRNA target in the cytoplasm and to block gene expression by binding and inactivating selected RNA sequences.
While the potential of antisense is widely recognized, there are numerous limitations to the use of antisense currently available. One of the key limiting aspects of this strategy is poor cell penetration. Akhtar et al., Nucleic Acids Res. 19:5551 (1991).
Morpholino oligonucleotides (also referred to herein as “morpholinos”) are oligonucleotides that include an antisense oligonucleotide and morpholine backbone. These antisense morpholinos, typically 18-25 nucleotides in length, can be designed to bind to a complementary sequence in a selected mRNA. The binding of the morpholino to the “target sequence” prevents translation of that specific mRNA, thereby preventing the protein product from being made. Morpholinos function by an RNase H-independent mechanism (i.e., a steric block mechanism as opposed to an RNase H-cleavage mechanism), and are soluble in aqueous solutions, with most being freely soluble at mM concentrations (typically 10 mg/ml to over 100 mg/ml). Nasevicius et al., Nat. Genet 26:216-220 (2000); Lewis et al., Development 128:3485-95 (2001); Mang'era et al., Eur. J. Nucl. Med. 28:1682-1689 (2001); Satou et al., Genesis 30:103-06 (2001); Tawk et al., Genesis 32:27-31 (2002); Lebedeva et al., Annu. Rev. Pharmacol. Toxicol. 4:403-19 (2001).
Morpholinos have numerous, significant advantages over the alternative phosphorothioates, which have been documented with a number of non-antisense effects. Morpholinos generally are stable in cells because their morpholine backbone is not recognized by nucleases. In addition, morpholinos are highly effective with predictable targeting, as compared to other antisense molecules. Nasevicius et al., Nat. Genet 26:216-220 (2000); Lewis et al., Development 128:3485-95 (2001); Mang'era et al., Eur. J. Nucl. Med. 28:1682-1689 (2001); Satou et al., Genesis 30:103-06 (2001); Tawk et al., Genesis 32:27-31 (2002); Lebedeva et al., Annu. Rev. Pharmacol. Toxicol. 4:403-19 (2001).
Key parameters for antisense inhibition by antisense oligodeoxiribonucleotides are their intracellular delivery and concentration. At the present time, it is believed that naked oligonucleotides enter the cell via active processes of adsorptive endocytosis and pinocytosis. However, the penetration of the endosomal barrier is a pre-requisite event for antisense activity and the naked antisense oligonucleotides do not appear to do this in great extent. Lebedeva et al., Annu. Rev. Pharmacol. Toxicol. 4:403-19 (2001); Weiss et al., Neurochem. Int. 31:321-48 (1997). Although complexes of antisense oligonucleotides with cationic liposomes, in some instances, have enhanced intracellular delivery, they have come with a disadvantage, cytotoxicity. Their utility in vitro and in vivo has also been limited by their lack of stability in serum and their inflammatory properties.
Conventional methods for the delivery of morpholinos in vitro include scrape loading and the so-called “special delivery vehicles.” Scrape loading entails adding oligonucleotides to adherent cells and scraping the cells from their plate, which disrupts the cell membrane temporarily allowing the oligonucleotide to enter the cell cytoplasm. Scraping the cells causes damage to the membrane, thereby reducing the viability of the cell population and ultimately altering the cellular characteristics of the remaining viable cells. Of the cells that do survive, not all may have received the morpholino. The second method, the “special delivery vehicle” supplied with the morpholino, requires dramatic changes in pH that result in very low efficacy. The low efficacy of the “special delivery vehicle” may be due to cytotoxicity or other changes to the cells.
The above methods are not translatable to in vivo delivery because they involve compromise of the target cells and pH changes. Furthermore, any in vivo delivery method or product must deliver the oligonucleotide to the cytosol. Without delivery to the cytosol, oligonucleotides remain trapped in the endosome/lysosome, or may be exocytosed.